Project Summary/Abstract In organisms spanning microbes to humans, the vast majority of cells have exited the cell division cycle and exist in a non-proliferative state. For many of these cells this is a reversible state of cell growth and cell cycle arrest known as quiescence. Quiescent cells are characterized by low metabolic, gene expression, and cellular activity. Quiescent cells are found in diverse clinical scenarios ranging from pathogenic microbes to human tumors. As these cells are not actively cycling, many drugs that target cellular processes such as DNA replication and protein synthesis are ineffective. The central hypothesis of our research proposal is that large- scale remodeling of protein expression in quiescent cells is mediated by signaling networks that respond to distinct signals to establish a common gene expression state. We postulate that this process leads to fundamental changes in the cytoplasm of the cell resulting in distinct biophysical properties that can benefit the long term survival of quiescent cells, and which can be exploited for therapeutic targeting of quiescent cells. The goal of this proposal is to test this hypothesis using the model eukaryotic cell, Saccharomyces cerevisiae. Our first aim is to define the dynamics with which the proteome is remodeled in response to distinct signals that initiate quiescence. Using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry, we will quantify the dynamics of protein expression changes in response to three different starvation signals that result in the initiation of quiescence: nitrogen, carbon and phosphorus. To test the role of specific signaling pathways in regulating remodeling of the proteome we will quantify expression dynamics in strains impaired for the TORC1, AMPK, PKA and PHO85 pathways as well as the signal integrators, RIM15 and SCH9. In aim 2 we will define the biophysical properties of quiescent cells. Using genetically encoded multimeric nanoparticles (GEMS) and imaging in microfluidics we will study cells as they enter quiescence and quantify changes in cytoplasmic diffusion to quantify cytoplasmic crowding. To identify factors that contribute to the altered biophysical properties of the cell we will quantify changes in organelle size and the abundance of macromolecular complexes such as the ribosome. We will use genetic and chemical perturbations to test their role in modulating the properties of quiescent cells and test the hypothesis that increased molecular crowding confers increased stress resistance. To identify effective therapeutic strategies for quiescent cells we will test the effectiveness of existing antifungal drugs in killing quiescent cells, identify genetic liabilities that enhance the efficacy of antifungals in quiescent cells and test the use of adjuvants that mimic genetic effects. To determine the clinical relevance of these findings, we will test newly identified therapeutic strategies in the pathogenic fungi, Candida albicans and Candida glabrata. Our study will provide a comprehensive understanding of how cells remodel their proteome to establish quiescence, its consequences for the biophysical properties of the cell, and therapeutic strategies for combating quiescent cells.